The search for a natural opiate receptor agonist in the brain led to the isolation of a low molecular weight peptide called "enkephalin" and to its identification by Hughes et al. as a mixture of two pentapeptides: H--Tyr--Gly--Gly--Phe--Met--OH (methionine enkephalin) and H--Tyr--Gly--Gly--Phe--Leu--OH (leucine enkephalin). The opiate receptor agonist properties of these pentapeptides were demonstrated by their ability to block electrically evoked contractions of mouse vas deferens and guinea pig ileum and by their ability to inhibit the stereospecific receptor binding of the opiate antagonist 3H-naloxone in brain homogenates, which are standard opiate receptor agonist properties of morphine.
It has been proposed that enkephalin receptors in the brain may be sites at which morphine-like drugs exert their analgesic effects. It is further proposed that the enkephalins may be the natural modulators or transmitters in the brain's systems for pain suppression or analgesia. The analgesic effect of methionine-enkephalin (Met-enkephalin) and leucine-enkephalin (Leu-enkephalin) was, in deed, reported by Belluzzi et al. in Nature, 260, 625-6 (1976). Their measurements of the analgesic effects of the two enkephalins injected intraventricularly into the rat brain in the tail-flick procedure showed that they produced analgesia equivalent to morphine, albeit at doses of 10-20 times that of morphine and for short intervals (10-12 minutes compared to an hour or more for morphine). Belluzzi et al. also found this analgesia to be reversible by the administration of naloxone, further demonstrating the opiate-like analgesic properties of the two natural enkephalins.
However, Met-enkephalin and Leu-enkephalin are virtually inactive when administered systemically (e.g. subcutaneously or intravenously), as reported by Frederickson et al. in Opiates and Endogenous Opioid Peptides, H. Kosterlitz, Ed., pp. 239-246, Elsevier/North-Holland, Biomedical Press, Amsterdam (1976). Strong evidence indicates that the reason for this lack of systemic activity and the short duration of intraventricular activity of the two enkephalins is the rapid enzymatic breakdown of the two peptides, particular at the Tyr.sup.1 --Gly.sup.2 peptide bond. For example, Chang et al., Life Sciences, 18, 1473 (1976), demonstrated that preincubation of the enkephalins at 34.degree. C. with brain membrane preparations results in a 50-100 percent loss of potency in the receptor assay. C. B. Pert et al., Kosterlitz, supra, at pp. 79-86, found that substitution of D-Ala.sup.2 for Gly.sup.2 of Met-enkephalin protected against degradation and produced a more potent, long-acting analgesic. Additionally, Hambrook et al., Nature, 262, 782-783 (1976), found that Met-enkephalin and Leu-enkephalin were, in fact, broken down in both rat and human blood plasma. It has also been suggested that poor transportation of the enkephalins across the blood-brain barrier may be partially responsible for their lack of analgesic activity after systemic administration.
Of further significance to the physiology of the enkephalins is the observation that Met-enkephalin has the same peptide sequence as the N-terminal portion of .alpha.-, .delta.-, and .beta.-endorphin, which, in turn, corresponds to the C-terminal sequence (61-91) of .beta.-lipotropin. .beta.-Lipotropin is found in large concentrations in the pituitary gland and in much lower concentrations in the brain. This relationship--coupled with the finding that the endorphins and .beta.-lipotropin also exhibit morphine-like properties in various test systems--has led to the suggestion that Met-enkephalin is a natural breakdown product of .beta.-lipotropin.
The various reports presented in Opiates and Endogenous Opioid Peptides, Kosterlitz, Ed., supra, are reviewed by Iverson and Dingledine in Nature, 262, 738-739 (1976). The role of the enkephalins in neurotransmission and analgesia is examined by Robert C. A. Frederickson in "Enkephalin Peptapeptides--a review of current evidence for a physiological role in vertebrate neurotransmission", Life Sciences, 21, 23-42 (1977). In a 1978 review, "Endogenous peptides and analgesia", Ann. Rev. Pharmacol. Toxicol., 18, 189-204, Lars Terenius further examines the endorphins (including the enkephalins) and pain mechanisms.
In order to obtain an enkephalin-like pentapeptide with analgesic activity upon peripheral (e.g. subcutaneous, intravenous, or oral) administration, numerous analogs have been made in which one or more of the amino acids in enkephalin have been replaced by another amino acid, removed, or structurally modified. For example, Belgian Pat. No. 853,448 to Imperial Chemical Industries, Limited, shows the compound (D-Met.sup.2, Leu.sup.5) enkephalin, methyl ester. U.S. Pat. No. 4,128,541 to Sarantakis discloses (Ser.sup.5)-enkephalin eliciting analgesic activity upon administration in the rat lateral brain ventricle. Dutta et al., Life Sciences, 21, 559-562 (1977), report that (D-Met.sup.2, Leu.sup.5) enkephalin, methyl ester shows strong opiate receptor agonist activity in the electrically stimulated guinea pig ileum model. Dutta et al., Id., at p. 560, also point out the lack of correlation between the standard in vitro opiate-receptor binding assays and the in vivo analgesic activity exhibited by some 200 enkephalin analogs which they investigated. Roemer et al., Nature, 268, 547-549 (1977), report that (D-Ala.sup.2, Met(O).sup.5)-enkephalin and (D-Ala.sup.2, Me-Phe.sup.4, Met(0).sup.5)-enkephalin elicited analgesic activity after oral administration. In separate papers, S. Bajusz and J. I. Szekely and their co-workers report that (D-Met.sup.2, Pro.sup.5)-enkephalin amide elicits analgesic activity when administered intracerebroventricularly, intravenously, or subcutaneously. [See Bajusz et al., Acta Biochem. et Biophys. Acad. Sci. Hung., II(1), 305-309 (1976) and Szekely et al., European Journal of Pharmacology, 43, 293-294 (1977)]. This compound is also disclosed in Derwent Abstract No. 19638A which is an abstract of Belgian Pat. No. 858,453 to Richter Gedeon Vegy.